In vivo immunodetection of thrombi and fibrin deposits remains an important clinical problem. In humans, the detection and localization of deep vein thrombi and coronary artery thrombi are two clinically important problems.
Blood clots when thrombin cleaves two pairs of small peptides from fibrinogen to yield fibrin monomers (Blomback, B. and Vestermark, A., Ark. Kemi 12: 173 (1958) and Doolittle, R. F., Adv. Protein Chem. 27: 1 (1973)). Fibrin monomers spontaneously aggregate to form an insoluble gel, which is then covalently stabilized by Factor XIIIa. Despite the dramatic differences between the two, fibrin retains 98% of the original covalent structure of fibrinogen. Thus, it is understandable that anti-fibrin serum cross-reacts strongly with fibrinogen, and that only one instance is known where a fibrin-specific serum was produced (Bosnjakovic, V. B. et al., Lancet 2: 452 (1977)).
Polyclonal antibodies raised against fibrin or fibrinogen have been utilized for localizing a variety of tumors, in particular the fast-growing kind (Day, E. D. et al., J. Natl. Cancer Insti. 22: 413 (1958), Bale, W. F. et al., Cancer Res. 20: 1488 (1960), Pressman, D., Cancer Res. 40: 2965 (1980)). However, these antibodies all showed to some extent cross-reactivity with fibrinogen.
Polyclonal antibodies to fibrin-fibrinogen, to fibrinogen alone or to degradation products of both polypetides have been developed for use in the detection of venous thrombosis in humans (Reich, T. et al., Surgery 60(6): 1211, Spar, I. L. et al., Circulation Research, XVII: 322 (1965), and Stephan, W. et al., U.S. Pat. No. 4,147,765). In Reich, T. et al. and Spar, I. L. et al., supra, the radiolabelled antibodies had a substantial degree of fibrin-fibrinogen cross-reactivity. In the Stephan, W. et al. reference, the antiserum was elicited by immunological challenge with the early fibrin and fibrinogen degradation products formed by the action of plasmin on fibrin and fibrinogen, and referred to in the literature as fibrinogen S (fg-X), and fibrinogen Y (fg-Y).
A different approach for the detection of deep-vein thrombosis was undertaken by Knight, L. C. et al., J. Nuclear Med. 19(8): 891 (1978) by radio-iodinating fibrinogen. This method, however, was found to be inferior to the labelling of platelets with .sup.111 In, for the localization of thrombi in deep-vein thrombosis.
When plasminogen is converted by an activator to plasmin, the active fibrinolytic enzyme of plasma, it develops a marked affinity for its substrate, fibrin. Ouchi, H. and Warren, R., Surgery 51(1): 42 (1962), discovered that this affinity might be put to use by tagging plasminogen with a radioactive isotope and using it as a tracer to seek out intravascular clots. However, the .sup.131 I-labelled plasminogen showed a somewhat depressed fibrinolytic activity (28.6%).
Pilgeram, L., U.S. Pat. No. 4,245,040 improved on an earlier method for detecting circulating fibrin (Lorand, L., in Fibrinogen and Fibrin Turnover of Clotting Factors, Ed. Koller F., F. K. Schattauer Verlag-Stuttgart, 1963; and Kisker, C. D. and Rush, R., in "Detection of Intravascular Clotting," J. Clin. Investig. 50:2235 (1971)). Pilgeram's improvement consisted in attaching an isotopic label to the soluble circulating fibrin, by means of a Factor XIII-containing glycine-C.sup.14 ethyl ester. However, it is recognized that the use of antifibrin antibodies which cross-react with fibrinogen is limited.
Monoclonal antibodies specific against fibrin have been described in Hui et al., Science, 222: 1129 (1983). Further description of the same type of antibodies can be found in commonly assigned co-pending U.S. application, Ser. No. 824,228, filed January 30, 1986, for "Fibrin-Specific Monoclonal Antibodies Lacking Fibrinogen Cross-Reactivity." Other examples of antibodies with a specificity against a thrombus include Kudryk et al., Mol. Imm., 21: 89 (1984); European Patent Application 146,050 to Callewaert, published June 26, 1985, for "Site Selective Plasminogen Activator and Method of Making and Using Same;" and Australian Patent Application, AV-A-25387/84 to Bundesen et al., for "Monoclonal Antibodies with Specificity for Crosslinked Fibrin and Their Diagnostic Uses."
A need continues to exist for highly specific antifibrin monoclonal antibodies and for synthetic epitopic peptides capable of raising such nonfibrinogen-cross-reacting antibodies. The antibodies would be useful for the in vivo detection of thrombi.